A synthetic polypeptide epitope based vaccine composition

ABSTRACT

Conserved epitopes selected from EV71 and CVA16, the two major causative agents of Hand Foot and Mouth Disease has been used to develop sub-unit bivalent vaccine antigen construct. The said vaccine described in this invention is capable to provide cross-protection to diverse EV71 and CVA16 infection causing strains. Further disclosed are the expression of the multi-epitope vaccine antigen coding gene and the purification process involved thereof. This present invention also discloses vaccine formulations against Hand Foot and Mouth Disease and other enterovirus infections comprising the recombinant vaccine antigen construct of the present invention.

FIELD OF THE INVENTION

The invention relates to a synthetic polypeptide epitope based vaccinecomposition. More particularly, conserved epitopes selected from EV71and CVA16 are used to develop a sub-unit bivalent vaccine antigenconstruct and a vaccine composition is provided using the developedsub-unit bivalent vaccine antigen construct, wherein the vaccine iscapable to provide cross protection to diverse EV71 and CVA16 infectioncausing strains. Further the expression of multi-epitope vaccine antigencoding gene and the purification process involved thereof are disclosed.The invention also discloses vaccine formulations against Hand Foot andMouth Disease and other enterovirus infections comprising therecombinant vaccine antigen construct of the present invention.

BACKGROUND OF THE INVENTION

Hand foot and mouth disease (HFMD) is a common pediatric disease causedpredominantly by Enterovirus-71 (EV71) and coxsackievirus A16 (CVA16).Both of them are single stranded positive-sense RNA viruses and belongto Picornaviridae family. EV71 also causes numerous neurologicalcomplications ranging from aseptic meningitis to acute flaccidparalysis, brain-stem encephalitis and even death. Often these twoviruses co-circulate and cause co-infection bringing devastating impacton healthcare system of many Asian countries.

EV71 and CVA16 are the two major etiological agents against Hand Footand mouth Disease. Both of the viruses are highly polymorphic andantibody(ies) against one will not provide significant cross-protectionagainst the other. Thus, a probable bivalent protein composition againstthese two viruses immense promise to reduce the global burden of thisdisease. It is well known that majority of the neutralizing antibody islocated in major capsid protein VP1 among enteroviruses. VP1 has lot ofsequence variability among different strains due to frequent mutationsand recombination events (Leitch E C M et al. J. Virol. 2012;86:2676-2685). Based on the VP1 sequence variation, EV71 has beenclassified so far into seven genogroups (GgA-GgG) and genogroups B and Care further subdivided into B1-B5 and C1-C5 (Brown B A et al. J. Virol.1999; 73:9969-9975).

Thus, development of vaccine against both of these viruses is highlydesirable to constrain them. The inventors of this present inventionproposes and hereby has developed a vaccine directed mainly against EV71and CA16 with potentiality to cross protect against other closelycirculating HFMD causing serogroups. An unique synthetic gene encodingmultiple copies of epitopes derived from capsid protein (VP1) fromEnterovirus-71 and Coxsackievirus-A16 have been designed and constructedto be used as a distinct and highly effective recombinant proteinconstruct with potential for immunization as a vaccine candidate againsthand foot and mouth infections caused by EV71 and CA16.

Further, the inventors have also developed and disclosed in thisinvention another vaccine candidate that encompass epitopes regions fromthe major capsid proteins VP1, VP2 and VP3 of EV71 and CVA16.

Objective of the Invention

In one object, the invention provides a synthetic polypeptide epitopebased vaccine composition.

In another object, the invention provides a recombinant sub-unitbivalent vaccine antigen construct using conserved epitopes selectedfrom EV71 and CVA16.

In another object, the invention provides a vaccine which is capable toprovide cross-protection to diverse EV71 and CVA16 infection causingstrains.

In another object, the invention further provides the expression of themulti-epitope vaccine antigen coding gene and the purification processinvolved thereof.

In another object, the invention provides vaccine formulations againstHand Foot and Mouth Disease and other enterovirus infections comprisingthe recombinant vaccine antigen construct of the present invention.

SUMMARY OF THE INVENTION

According to one embodiment of the invention, the schematic structureand the codon optimized genetic and protein sequences of multi-epitopebased vaccine antigens against hand foot and mouth disease is disclosed.

According to another embodiment of the invention, the expression ofcodon optimized sequences of the present invention as inclusion bodiesin appropriate host is disclosed.

Further embodiment of the invention describes the method of proteinpurification comprising lysis with lysis buffer-I and lysis buffer-II,washing with wash buffers I, II, and III, Immobilized metal affinitychromatography followed by dialysis and refolding and recovery of theproteins of interest of SEQ ID No. 2 and SEQ ID No. 4 with appropriaterefolding buffers, subsequently followed by specific chromatographicpurification techniques such as size exclusion chromatography.

The further embodiments of the invention also establish immunogenicityof the invention through appropriate animal studies. The SEQ ID Nos. 2and 4 is capable to generate sufficient immune response against HandFoot and Mouth disease caused by enterovirus and coxsackievirus. Thevaccine antigens of the present invention are also capable to inducecross-protection against any strains of enterovirus and coxackieviruscausing hand foot and mouth disease in humans.

Specific vaccine formulations comprising SEQ ID No. 2 and SEQ ID No. 4with multiple adjuvants have also been made available as one of theembodiments of the present invention optionally in presence of otherstabilizers like polyols, sugar or amino acids or combinations.

In another aspect of the instant invention there is provided a vaccinecomposition for prophylaxis against Hand Foot and Mouth Disease causedby EV71 and CA16 comprising: (a) vaccine antigen, the said vaccineantigen is a synthetic construct selected from the recombinant proteinsequences as represented by SEQ ID No. 2 (named as MEV1) and SEQ ID No.4 (named as MEV2); (b) adjuvants; (c) stabilizers; and (d) anyphysiologically acceptable buffer selected from phosphate, and citrate,wherein the said vaccine formulation is stable for at least 2 years at5±3° C. and up to 2 weeks at 37° C.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Multi-epitope bivalent vaccine gene construct againstEnterovirus A71 and Coxsackievirus A16 (MEV1 and MEV2).

1A: The schematic representation of MEV1 showed that four copies of thethree epitopes are present in tandem. Linker separates the epitopes toadd flexibility. NdeI and BamHI sequences are present at the 5′ and 3′end respectively.1B: The schematic representation of MEV2 showed that three copies of sixepitopes are present in tandem followed by one copy of three moreepitopes. Linker separates the epitopes to add flexibility. NdeI andBamHI sequences are present at the 5′ and 3′ end respectively.

FIG. 2: Different concentration of IPTG has been evaluated to assess theoptimum concentration for expression of MEV1 (2A) and MEV2 (2B). 2A:From left Lane 1: Un-induced cells (in absence of IPTG), Lane2-4: Cellsinduced with 0.2 mM, 0.5 mM and 0.75 mM IPTG respectively, Lane 5:Molecular weight marker for protein with sizes 16 kDa, 29 kDa, 33 kDa,43 kDa, 54 kDa, 71 kDa, 91 kDa, 124 kDa and 250 kDa respectively frombottom, Lane 6-8: Cell pellet with protein aggregates after induced with0.2 mM, 0.5 mM and 0.75 mM IPTG respectively and followed by lysis, Lane9-11: Cell lysate after induced with 0.2 mM, 0.5 mM and 0.75 mM IPTGrespectively and followed by lysis.

2B: From Left, Lane1: Un-induced BL21 (DE3) cells transformed withpET11b-MEV2 (in absence of IPTG), Lane 2-4: Induction of BL21 (DE3)cells transformed with pET11b-MEV2 using 0.2 mM, 0.5 mM and 0.75 mM IPTGConclusion: The results from the figure showed that expression of MEV1and MEV2 can be induced using wide range of IPTG concentration.

FIG. 3: The level of expression of MEV1 (3A, 3B) and MEV2 (3C) isevaluated in different temperature settings.

(3A):—From left Lane 1: Un-induced cells (in absence of IPTG), Lane 2:Molecular weight protein marker as described in FIG. 1, Lane 3-4: Celllysate and cell pellet respectively after induced with 0.2 mM IPTG at37° C. and followed by lysis, Lane 4-5: Cell lysate and cell pelletrespectively after induced with 0.2 mM IPTG at 25° C. and followed bylysis.(3B):—From left Lane 1: Un-induced cells (in absence of IPTG, Lane 2:Cells induced with IPTG at 37° C., Lane 3-4: Cell pellet (P) and celllysate (S) respectively after induced with 0.2 mM IPTG at 37° C. andfollowed by lysis, Lane 5: Cells induced with IPTG at 25° C., Lanes 6-7:Cell pellet (P) and cell lysates (S) respectively after induced with 0.2mM IPTG at 25° C. and followed by lysis, Lane 8: Molecular weightprotein marker as described in FIG. 1.(3C): From left Lane1: Un-induced MEV2 gene transformed Rosetta cells(in absence of IPTG), Lane 2: Rosetta cells induced with 0.2 mM IPTG at18° C., Lane 3: Rosetta cells induced with 0.2 mM IPTG at 37° C., Lane4:Un-induced MEV2 gene transformed BL21 (DE3) cells, Lane 5: BL21 (DE3)cells induced with 0.2 mM IPTG at 37° C., Lane 6: BL21 (DE3) cellsinduced with 0.2 mM IPTG at 18° C., Lane 7: Molecular weight proteinmarker.Conclusion: The results obtained from FIGS. 3A and 3B depicted that 37°C. is the optimum temperature to induce MEV1 and MEV2 by IPTG.

FIG. 4: Two different concentrations of urea (6M and 3M) were evaluatedfor improving the solubility of MEV1 (4A) while for MEV2, solubility wasalso assessed with two different concentrations of urea (6M and 3M) inpresence or absence of DTT with variable pH (4B, 4C).

4A: From left Lane 1: Cell lysate after induced with 0.2 mM IPTG andfollowed by lysis, Lane 2: Supernatant after insoluble cell aggregateswas treated with 6M urea, Lane 3: Cell pellet after insoluble cellaggregates was treated with 6M urea, Lane 4: Supernatant after insolublecell aggregates was treated with 3M urea, Lane 5: Cell pellet afterinsoluble cell aggregates was treated with 3M urea, Lane 6: Molecularweight protein marker as described in FIG. 1.4B: From left, Lane1: Supernatant after insoluble cell aggregates wastreated with 3M urea, Lane 2: Cell pellet after insoluble cellaggregates was treated with 3M urea, Lane 3: Supernatant after insolublecell aggregates was treated with 3M urea in presence of 10 mM DTT, Lane4: Cell pellet after insoluble cell aggregates was treated with 3M ureain presence of 10 mM DTT, Lane 5: Supernatant after insoluble cellaggregates was treated with 6M urea, Lane 6: Cell pellet after insolublecell aggregates was treated with 6M urea, Lane 7: Supernatant afterinsoluble cell aggregates was treated with 6M urea in presence of 10 mMDTT, Lane 8: Cell pellet after insoluble cell aggregates was treatedwith 6M urea in presence of 10 mM DTT, Lane 9: Molecular weight proteinmarker as described in FIG. 2B.4C: From Lane 1: Cell lysate after induced with 0.2 mM IPTG and followedby lysis in Lysis buffer I (ph7.4), Lane 2: supernatant after insolublecell aggregates was treated with 6M urea in lysis buffer II (ph7.4) inpresence of 20 mM DTT, Lane 3: cell pellet after insoluble cellaggregates was treated with 6M urea in Lysis buffer II (ph7.4) inpresence of 20 mM DTT, Lane 4: Cell lysate after induced with 0.2 mMIPTG and followed by lysis in lysis buffer (50 mM Na₂HPO₄, 0.3M NaCl, 1%TritonX-114, 0.5 mg/ml lysozyme, 1 mM AEBSF, at pH 8), Lane 5:supernatant after insoluble cell aggregates was treated with 6M urea inlysis buffer 50 mM Na₂HPO₄, 0.3M NaCl, 1% TritonX-114, 0.5 mg/mllysozyme, 1 mM AEBSF, at pH 8) in presence of 20 mM DTT, Lane 6: Cellpellet after insoluble cell aggregates was treated with 6M urea in lysisbuffer 50 mM Na₂HPO₄, 0.3M NaCl, 1% TritonX-114, 0.5 mg/ml lysozyme, 1mM AEBSF, at pH 8) in presence of 20 mM DTT, Lane 7: Molecular weightprotein marker as described in FIG. 2B, Lane 8: Cell lysate afterinduced with 0.2 mM IPTG and followed by lysis in lysis buffer (50 mMTris; 0.3 M NaCl, 1% TritonX-114, 0.5 mg/ml lysozyme, 1 mM AEBSF, at pH8.5), Lane 9: supernatant after insoluble cell aggregates was treatedwith 6M urea in lysis buffer (50 mM Tris, 0.3M NaCl, 1% TritonX-114, 0.5mg/ml lysozyme, 1 mM AEBSF, at pH 8.5) in presence of 20 mM DTT, Lane10: Cell pellet after insoluble cell aggregates was treated with 6M ureain lysis buffer (50 mM Tris, 0.3M NaCl, 1% TritonX-114, 0.5 mg/mllysozyme, 1 mM AEBSF, at pH 8.5) in presence of 20 mM DTT (The volume ofcell pellet was very less for Lane 10 sample as it got solubilizedwell).Conclusion: The figure concludes that the 3M and 6M urea treatment canbe recovered with significant amounts of MEV1 with higher purity whencompared to the untreated samples whereas presence of 6M urea and DTT isrequired to achieve significant amount of soluble MEV2 protein.

FIG. 5: Expression and purification of EV-Ag (MEV1).

(5A): Expressed MEV1 was purified by affinity chromatography with IMACtechnology using Ni-NTA resins and SDS-PAGE electrophoresis wasperformed). From left Lane 1: Un-induced cell pellet (in absence ofIPTG), Lane 2: Supernatant from inclusion bodies (IB) like cellaggregates treated with 3M urea, Lane 3: Purified protein after IMACmediated purification using N-NTA resin, Lane 4: Molecular weightprotein marker as described in FIG. 1.(5B): The specific expression of MEV1 was detected by Western Blot usinganti-his antibody. From left Lane 1: (UP)—Unpurified protein insupernatant derived from inclusion bodies (IB) like cell aggregatesafter treatment with 3M urea, Lane 2: (P)—Purified protein after IMACmediated purification using Ni-NTA resin.Conclusion: The results from FIG. 5 showed that the IMAC purificationstep generated more than 90% purified MEV1 and the specific expressionwas confirmed by western blotting using anti-his antibody.

FIG. 6: IgG1/IgG2 a ratio was determined for the MEV1 immunized Balb/Cmice sera (1:250) in presence of different adjuvants. CFA/IFA: Miceimmunized with MEV1 in presence of Complete Freud's adjuvant as primedose and MEV1 in presence of Incomplete Freud's adjuvant as subsequentbooster dose, Alum: Mice immunized with MEV1 in presence of Alum asadjuvant, MPLA+ Alum: Mice immunized with MEV formulated with Alum andMPLA both. Poly(I:C): Mice immunized with MEV formulated with Poly(I:C),AddaVax: Mice immunized with MEV formulated with AddaVax. MEV1 coatedELISA plates were treated with the sera (1:250) and finally incubatedwith either anti-mouse IgG1-HRP or IgG2a-HRP secondary antibody.

Conclusion: CFA/IF And Alum adjuvanted MEV1 immunization generated muchhigher IgG1 antibody indicating predominant Th2 type immune responsewhile Poly(I:C), AddaVax and MPLA+ Alum generated slightly higher IgG2aantibody indicating overall balanced Th1/Th2 response with slight biasfor Th1 type immune response.

FIG. 7: Binding analysis of MEV1 immunized sera with EV71 and CVA16 byImmunofluorescence. Sera were collected from mice immunized with MEV1formulated with Alum or from mice immunized with PBS formulated withalum. Subsequently, Vero cells were infected with either Enterovirus A71(EV71) and Coxsackievirus A16 (CVA16) at 0.01 Multiplicity of Infection.Infected Vero cells were treated with 1:200 diluted sera as mentioned inthe corresponding panel of the figure followed by incubation with Alexa488 conjugated anti-mouse IgG secondary antibody and observed underfluorescence microscope. Bright field: Bright field of the microscope,Fluorescence: Green fluorescent field of the microscope, Ag+ Alum: Seracollected from mice immunized with MEV1 formulated with Alum, PBS+ Alum:Sera collected from mice immunized with PBS formulated with Alum(negative control sera), EV71 infected Vero cells: Panels where Verocells infected with EV71 has been used, CVA16 infected Vero cells:Panels where Vero cells infected with CVA16 has been used, Mock infectedVero cells: Panels where Vero cells were not infected with virus(negative control for infected Vero cells). Conclusion:Immunofluorescence result showed the cross-reactivity of the antibodygenerated against both EV71 and CVA16.

FIG. 8: IgG Titer of the serum from MEV-1 antigen immunized mice inpresence of different Adjuvants. Serum was collected two-weeks after the2^(nd) boost. ELISA was performed in the antigen coated ELISA plate inpresence of different concentration of serum following blocking.Subsequently, Secondary IgG-HRP conjugated antibody was added. TMBsubstrate was added for colour generation which was stopped with 0.6-1MHCL. The color intensity was recorded at 450 nm in an ELISA reader.

FIG. 9: Quantitation of IFN-γ (9A) and IL-4 (9B) secreted fromsplenocytes of mice immunized with MEV1 formulated with differentadjuvants.

FIG. 10: Stability of the synthetic protein construct MEV-1, from Left,Lane 1: MEV1 protein without stabilizer at 37±12° C. for 2 weeks, Lane2: MEV1 protein in 20% Glycerol at 37±12° C. for 2 weeks, Lane 3: MEV1protein not exposed to 37±12° C. (control protein), Lane 4: Molecularweight protein marker.

DETAILED DESCRIPTION OF THE INVENTION

In the present invention, four copies of each epitope was connected bylinkers to construct the recombinant antigen Multi-epitope Enterovirusantigen 1 (MEV1) that functions as a vaccine antigen gene construct. Thetarget gene (MEV1) as disclosed in SEQ ID No.1 and the resultant proteinas disclosed in SEQ ID No. 2 is designed to possess four copies of eachof one enterovirus A71 epitope, one coxsackievirus A16 epitope and oneextremely conserved epitope having 100% homology to enterovirus A71strains and high homology to Coxsackievirus A16 and some otherCoxsackievirus strains and thus will provide cross protection. All theepitopes are present in VP1 capsid gene of either Enterovirus A71 orCoxsackievirus A16. The target gene with NdeI and BamH1 restrictionsites at 5′ and 3′ end respectively was used as insert to be introducedin NdeI-BamHI cloning site of the expression vector pET11b. The insertalso include C-terminal 6× histidine for easy detection and purificationof the expressed protein. The presence of poly-histidine tag enables theprotein to be purified by immobilized metal affinity chromatography inthe subsequent steps and also aids in the detection of the expressionusing poly-histidine specific antibody. 6× histidine tag has severaladvantages including smaller size, low toxicity and immunogenicity andthus doesn't interfere with the immunogenicity of the antigen. Thevector with the insert was transformed in the E. coli DH5α (specificallyfor cloning) and BL21 (DE3) cells for expression of the said geneconstruct of the present invention.

The present invention also includes another construct MEV2 that includesnine epitopes from VP1-3 proteins of EV71 CVA16 (MEV2). These epitopesincludes both B cell and T cell epitopes. The target gene (MEV2)sequence is disclosed in SEQ ID no 3 and the resultant protein sequenceis disclosed in SEQ ID no 4.

SEQ ID No 1: Nucleic acid sequence of MEV1CATATGGCTGCAGGTTCTGGTTACGACGGTTACCCGACCTTCGGTGAACACAAACAGGAAAAAGACCTGGAATACGGTGGTTCTGGTGGTTACCTGTTCAAAACCAACCCGAACTACAAAGGTAACGACATCAAAGGTGGTTCTGGTGGTATGCGTATGAAACACGTTCGTGCTTGGATACCGCGTATGCGTGGTGGTTCTGGTGGTTACGACGGTTACCCGACCTTCGGTGAACACAAACAGGAAAAAGACCTGGAATACGGTGGTTCTGGTGGTTACCTGTTCAAAACCAACCCGAACTACAAAGGTAACGACATCAAAGGTGGTTCTGGTGGTATGCGTATGAAACACGTTCGTGCTTGGATACCGCGTATGCGTGGTGGTTCTGGTGGTTACGACGGTTACCCGACCTTCGGTGAACACAAACAGGAAAAAGACCTGGAATACGGTGGTTCTGGTGGTTACCTGTTCAAAACCAACCCGAACTACAAAGGTAACGACATCAAAGGTGGTTCTGGTGGTATGCGTATGAAACACGTTCGTGCTTGGATACCGCGTATGCGTGGTGGTTCTGGTGGTTACGACGGTTACCCGACCTTCGGTGAACACAAACAGGAAAAAGACCTGGAATACGGTGGTTCTGGTGGTTACCTGTTCAAAACCAACCCGAACTACAAAGGTAACGACATCAAAGGTGGTTCTGGTGGTATGCGTATGAAACACGTTCGTGCTTGGATACCGCGTATGCGTCATCATCACCATCACCACTAAGGATCC(The location of Ndel and BamH1 sites has beenunderlined and the location of the 6X histidinehas been shown in bold in the actual sequences in SEQ ID No. 1 above) SEQ ID No 2: Amino acid sequence of MEV1MAAGSGYDGYPTFGEHKQEKDLEYGGSGGYLFKTNPNYKGNDIKGGSGGMRMKHVRAWIPRMRGGSGGYDGYPTFGEHKQEKDLEYGGSGGYLFKTNPNYKGNDIKGGSGGMRMKHVRAWIPRMRGGSGGYDGYPTFGEHKQEKDLEYGGSGGYLFKINPNYKGNDIKGGSGGMRMKHVRAWIPRMRGGSGGYDGYPTFGEHKQEKDLEYGGSGGYLFKTNPNYKGNDIKGGSGGMRMKHVRAWIPRMRH HHHHHSEQ ID No 3: Nucleic acid sequence of MEV2CATATGCGTCGTGGCAGCTATGATGGTTATCCGACCTTCGGCGAGCACAAACAAGAAAAAGACCTGGAATACGGCGGCGGCAGCGCGGGCGGCACCGGCACCGAGGACAGCCACCCGCCGTATAAACAAACCCAACCGGGTGCGGGTGGCGGTAGCGTGAACAACGTTCCGACCAACGCGACCAGCCTGATGGAGCGTCTGGGCGGTCCGGGCTACCCGACCTTCGGTGAACACCTGCAAGCGAACGACCTGGATTATGGCCAGTGCGGCGGTGGCAGCAACCAACCGTACCTGTTTAAAACCAACCCGAACTATAAGGGTAACGACATCAAAGGTGGCGGTAGCCACTACCGTGCGCACGCGCGTGCGGGTTATTTCGACTACTATACCGGTCCGGGTCCGTACGATGGCTATCCGACCTTTGGCGAGCACAAGCAGGAAAAAGACCTGGAGTATGGCGGTGGCAGCGCGGGTGGCACCGGCACCGAAGATAGCCACCCGCCGTACAAACAAACCCAGCCGGGTGCGGGTGGCGGTGGCAGCGTGAATAATGTGCCGACCAATGCGACCAGCCTGATGGAACGTCTGGGTGGCCCGGGCTATCCGACCTTTGGCGAACACCTGCAAGCGAATGACCTGGATTACGGCCAATGCGGCGGTGGCAGCAATCAGCCGTACCTGTTTAAGACCAATCCGAATTATAAGGGCAACGACATTAAAGGTGGCAGCCACTATCGTGCGCACGCGCGTGCGGGGTACTTTGACTACTATACCGGTCCGGGTCCGTACGATGGCTATCCGACGTTTGGTGAACACAAGCAGGAGAAAGACCTGGAATATGGTGGTGGTAGCGCGGGTGGCACCGGCACCGAGGATAGCCACCCGCCGTATAAACAAACGCAACCGGGTGCGGGCGGTGGCAGCGTGAATAATGTTCCTACTAATGCTACCAGCCTGATGGAACGCCTGGGTGGTCCGGGTTACCCGACTTTTGGCGAACACCTGCAAGCAAATGACCTGGATTATGGCCAATGCGGTGGCGGTAGCAATCAACCTTACCTGTTTAAGACTAACCCGAACTATAAGGGCAACGACATCAAAGGCGGTGGCAGCCACTATCGTGCGCACGCGCGTGCGGGCTATTTCGATTACTATACCGCGGGCGCGGGTGCGCAGCTGACCATCGGTAACAGCACCATTACCACCCAAGAAGCGGCGAACATCGGCGGTGGCAGCCCGCACCAGTGGATTAACCTGCGTACCAACAACTGCGCGACCATCATTGGTGGCGGTAGCATGGCGACCGGTAAAATGCTGATTGCGTACACCCCGCCGGGTGGTCCGCTGCCG TAAGGATCCSEQ ID No 4: Amino acid sequence of MEV2MRRGSYDGYPTFGEHKQEKDLEYGGGSAGGTGTEDSHPPYKQTQPGAGGGSVNNVPTNATSLMERLGGPGYPTFGEHLQANDLDYGQCGGGSNQPYLFKTNPNYKGNDIKGGGSHYRAHARAGYFDYYTGPGPYDGYPTFGEHKQEKDLEYGGGSAGGTGTEDSHPPYKQTQPGAGGGGSVNNVPTNATSLMERLGGPGYPTFGEHLQANDLDYGQCGGGSNQPYLFKTNPNYKGNDIKGGSHYRAHARAGYFDYYTGPGPYDGYPTFGEHKQEKDLEYGGGSAGGTGTEDSHPPYKQTQPGAGGGSVNNVPTNATSLMERLGGPGYPTFGEHLQANDLDYGQCGGGSNQPYLFKTNPNYKGNDIKGGGSHYRAHARAGYFDYYTAGAGAQLTIGNSTITTQEAANIGGGSPHQWINLRTNNCATIIGGGSMATGKMLIAYTPPGGPLP

Furthermore, the recombinant genetic constructs of the present inventionof the said vaccine antigen disclosed in this invention may comprise Bcell or T cell epitopes from any enterovirus including but not limitingto EV71, EVD68, Coxsackievirus A16, Coxsackievirus A4-6, CoxsackievirusA10, echoviruses etc. The multi-epitope construct(s) mentioned in thisinvention may also include carrier protein(s) for better immunogenicitythat may include any toxoids, TLR ligands like flagellin either as wholeprotein or truncated protein, CTL epitopes, T helper epitopes,immunomodulants, virus like particles etc. The antigen gene may alsoinclude one or more tags like poly-histidine tags, V5 tag, GST tag,signal sequences etc.

The proposed recombinant genetic constructs of the present invention canbe expressed in bacteria, yeast, mammalian cell or virus. The vector maybe plasmid or viral vector. In one embodiment, the expression system isEscherichia coli. Codon optimization is an essential step for successfulproduction of heterologous protein in E. coli. In the present invention,MEV1 and MEV2 gene have been codon optimized for successful productionin E. coli. In E. coli expression system, the heterologous protein oftenforms insoluble aggregates and remains inside the cells as inclusionbodies even after lysis with bacterial lysis buffer. Inclusion Body (IB)Proteins. All inclusion body proteins are highly specific in theirbio-chemical properties, Each inclusion body forming protein needsspecific experimental combinations such as specific combination of washbuffers for washing and cell lysis, denaturation components of theprotein followed by refolding in specific refolding buffers of the saidtarget protein for appropriate functional protein structure. Downstreamprocessing including cell lysis, denaturation and refolding of anyinclusion body forming protein generated from a synthetic recombinantgenetic construct is far more difficult and unpredictable since thenatural properties of the said protein are not at all known initially ascompared to those proteins already available in nature but produced asinclusion body forming proteins through human intervention.

In the present invention, phosphate buffer has been used for MEV1 as itis non-toxic and a common component of physiological fluids. Its pHalters little with temperature. It is colourless and thus doesn'tinterfere with light absorbance during protein quantitation. Further,NaCl is used to reduce the nonspecific hydrophobic protein interactions.Lysozyme was used as it enhances cell lysis by acting on bacterial cellwall peptidoglycan. In the present invention, freeze thaw cycles andsonication has been used for disruption of bacterial cell leading tocomplete lysis. Short pulse of sonication has been used with chilling inice in between to avoid high temperature generation that will degradethe protein. 4-(2-Aminoethyl) benzenesulfonyl fluoride (AEBSF) is abroad spectrum serine protease inhibitor and is non-toxic. AEBSF hasbeen used in this invention to prevent the serine protease mediateddegradation of MEV1. 4-(2-Aminoethyl)benzenesulfonyl fluoridehydrochloride or AEBSF is a serine-protease inhibitor that preventserine proteases mediated degradation of protein. In another embodimenteither phosphate buffer (ph7.4-8) or Tris buffer (ph8-8.5) have beenused for lysis and solubilization of MEV1 and MEV2. Tris lysis bufferconsists of 20-50 mM Tris, 0.3M NaCl, 1% TritonX-114, 0.5 mg/mllysozyme, 1 mM AEBSF, at ph8-8.5.

TritonX-114 is a mild non-ionic detergent. It is used in cell lysis. Italso has role in endotoxin reduction by phase separation at temperaturehigher than 20° C. Imidazole is a competitor for histidine to bind IMACresins. Thus, in low concentration, it prevents non-specific binding andthus added in wash buffers. In higher concentration (varies betweendifferent proteins), it will release the tagged proteins and the desiredprotein will come in the elutes. The function of these specificcomponents have been strategically optimized at each specific step thatwould best suit the present invention for these new proteins from thenovel genetic construct of the present invention MEV1, MEV2 or similarconstructs comprising enterovirus epitopes. These components areincluded as part of purification procedure in this present invention.

In general, the denaturating agents like urea or Guanidine HCl are usedto denature any IB protein. In this process, the protein will getdenatured and will come out in the solution. But, later protein can berenatured and refolded by stepwise or gradient decrease of ureaconcentration. However often, some specific additional components arerequired those technically assist in refolding and/or thereby preventthe aggregation of the varied category of IB proteins depending uponthere bonding and three dimensional structures. L-Arginine andL-Arginine HCl has been found to be essential in preventing theaggregation of MEV1 and MEV2 of this present invention. Reducing agentslike DTT or DTE is suitable for solubilizing MEV2.

The purification steps may involve affinity purification, size exclusionchromatography, ion exchange chromatography or any of the combinationsthereof. The buffer(s) used for washing and purification of therecombinant protein derived from the recombinant genetic construct inthis present invention includes phosphate buffer, Tris buffer, MOPSbuffer etc.

The recombinant protein(s) in this invention can be formulated withadjuvants that includes but not limited to alum (aluminium phosphate,aluminium hydroxide), squalene based adjuvants like MF59, montanide etc,RIBI adjuvants, Complete Freud's adjuvant and Incomplete freud'sadjuvant for immunogenicity testing, adjuvant involving bacterial cellcomponents or modified versions such as MPL, muramyl dipeptide etc, alloil-in-water emulsions, all water-in-oil emulsions, TLR ligand basedadjuvants, CpG and non CpG containing oligonucleotides, saponinsincluding but not limited to QS-1, ISCOM, ISCOMATRIX etc, vitamins,immunomodulants including cytokines.

The route of administration of the vaccine can be oral or parenteralwhere parenteral route includes but not limiting to intramuscular,intranasal, subcutaneous, tropical, intradermal or transdermal.

Example 1: Expression of the Recombinant Protein of the PresentInvention from MEV1 and MEV2 Genetic Construct

The plasmid pET11b and pET11b-MEV1 was transformed in E. coli BL21 (DE3)chemically competent cells. The transformed BL21 (DE3) cells wereincubated with shaking at 37° C. until OD₆₀₀ reaches 0.4 to 0.6, andthen induced with different concentration of Isopropylβ-D-1-thiogalactopyranoside (IPTG) at 37° C. for 4 hours. IPTG is amolecular mimic of a lactose metabolite that triggers the transcriptionof lac operon and thus induces the expression of the recombinant proteinwhere the gene is controlled by the lac operator. The optimumconcentration of IPTG for expression was determined and then theinduction of the multi-epitope gene (MEV1) for expression was alsodetermined at various temperature settings (FIG. 3). The optimumcondition for expression was assessed for future batches of experiments.pET11b-MEV2 was also transformed in BL21 (DE3) cells followed byincubation with shaking at 37° C. until OD₆₀₀ reaches 0.4 to 0.6, andthen induced with different concentration of Isopropylβ-D-1-thiogalactopyranoside (IPTG) at 37° C. for 4 hours. The optimumcondition for expression was also determined.

Example 2: Cell Lysis with Lysis Buffer-I and Lysis Buffer-II

The cell pellet of MEV1 transformed BL21 (DE3) after IPTG induction wascollected by centrifuging the cells at 10,000 rpm for 5 mins. The cellswere suspended in suspension/lysis buffer-I, the said lysis buffer-I wasprepared by making a solution containing 50 mM Na₂HPO₄, 0.3M NaCl, 1%TritonX-114, 0.5 mg/ml lysozyme, 1 mM AEBSF, at pH 7.4. Cell lysis wasperformed first by performing three rounds of freeze thaw cycles andsubsequently by ultrasonicating for 20 seconds thrice with 30 secinterval in between. The location of the desired protein was againchecked by SDS-PAGE. The majority of the desired protein was found incell debris indicating formation of insoluble inclusion/inclusion likebodies. The cells were then lysed in Lysis Buffer-II with differentconcentrations of urea (3-6M) in presence of 5-10 mM imidazole, 50 mMNa₂HPO₄, 0.3M NaCl, and 1 mM AEBSF at 4° C. for 3-16 hours to recoverthe insoluble protein.

Results showed that the recombinant enterovirus vaccine gene (MEV1) wasinduced best using 0.2 mM IPTG at 37° C. The protein was found to beexpressed ˜31 kDa region (FIG. 2) which coincide with the theoreticalmolecular weight of the protein calculated. To detect the extent oflysis, The SDS-PAGE analysis of the lysed cell free supernatant showedthat only ˜10% of the protein was present in the supernatant and therest in the cell pellet (FIG. 2). The level of expression of the desiredgene has not improved significantly at 25° C. (FIG. 3A) and at 15° C.(FIG. 3B) in comparison to that at 37° C. Thus, 37° C. has been selectedas optimum temperature for expression of MEV1. Then, the cells weretreated with 3M and 6M urea denaturation buffer to increase therecombinant protein recovery. It was found that up to ˜65% of theprotein can be recovered with ˜2-5% difference in protein recovery usingtwo different urea concentrations along with the increase of purity(FIG. 4 and Table 1). For the ease of renaturation, 3M ureaconcentration was selected to be used for later batches.

MEV-2 was also found to be expressed well in presence of 0.2-0.75 mMIPTG at ˜47 kDa region (FIG. 2B). MEV2 didn't express well at 15-18° C.The expression was only found at ˜37° C. MEV-2 expressed well whentransformed in both BL21 (DE3) and Rosetta cells. Like MEV1, MEV2 didn'tlyse well in presence of Lysis Buffer-I (FIG. 4C). Lysis Buffer-I forMEV2 comprised 50 mM Tris, 0.3 M NaCl, 1% Triton X114, 0.5 mg/mllysozyme, 1 mM AEBSF at pH 8-8.5. Unlike MEV-1, MEV-2 didn't lyse andsolubilize properly in presence of 3M urea. The solubility was slightlybetter in 6M urea. As, MEV2 protein sequence have four cysteinemolecules, we predicted the presence of disulphide bonds in MEV2 nativeprotein. Therefore, the addition of reducing agents like DTT can aid indisrupting the disulphide bond/s and thus will increase the solubility.We found that MEV2 protein get lysed and solubilized in lysis buffer-IIthat comprised 50 mM Tris, 0.3 M NaCl, 1 mM AEBSF in presence of 6M ureaand 10-20 mM DTT. No significant solubility has been observed inpresence of 3M urea and DTT at pH of 8-8.5.

Example 3: Immobilized Metal Affinity Chromatography (IMAC) and Washingof MEV 1 with Wash Buffer-I, II and Wash Buffer-III

The urea denatured protein solution in presence of 5-10 mM imidazole wasadded to the Ni-NTA IMAC columns after equilibrating with lysisbuffer-II and incubated for proper binding at 4° C. for 3-16 hrs. Thecolumn was then washed with Wash Buffer-I, the said Wash Buffer-I wasprepared by making a solution containing 50 mM Na₂HPO₄, 0.3M NaCl, 1 mMAEBSF, 3M urea at pH7.4 with 0.1% Triton X-114 and 5-10 mM Imidazole.Next wash was performed in Wash Buffer-II, the said Wash buffer-II wasprepared by making a solution containing the components 50 mM Na₂HPO₄,0.3M NaCl, 1 mM AEBSF, 3-6M urea at pH7.4 with 0.1% Triton X-114 and 20mM imidazole. Subsequently depending upon the initial concentration ofurea used in example 2, further at least two or more washes wereperformed with Wash Buffer-III in presence of gradual decreasing ureaconcentrations, the said Wash Buffer-III was prepared by making asolution containing 50 mM Na₂HPO₄, 0.3 M NaCl, 1 mM AEBSF additionallywith 20 mM imidazole with decreasing concentrations of urea. The saidWash buffer III did not contain TritonX-114 unlike Wash Buffer-II. Thetarget protein of interest of the present invention was finally washedin the column using Wash Buffer III containing 50 mM Na₂HPO₄, 0.3M NaCl,1 mM AEBSF under gradual decrease in concentration of urea andthereafter eluted from the column through an elution buffer 50 mMNa₂HPO₄, 0.3M NaCl, 1 mM AEBSF by increasing concentrations of imidazole(250-500 mM) with or without any urea at all.

Alternatively, during IMAC purification, washes are performed with WashBuffer-III containing 50 mM Na₂HPO₄, 0.3M NaCl, 1 mM AEBSF in presenceof 3-6M urea and subsequently the target protein of interest of thepresent invention is eluted from the column with an elution buffercomprising increasing concentration of imidazole (250-500 mM) inpresence of 3-6M urea, 50 mM Na₂HPO₄, 1 mM AEBSF, 0.3 M NaCl.

Example 4: Dialysis of the Target Protein MEV1 and MEV2 and SizeExclusion Chromatography and Protein Refolding

Ni-NTA column based affinity purified protein (MEV-1) was furtherdialyzed against PBS (phosphate buffered saline) in presence ofrefolding buffers comprising 0.1-0.3M NaCl, 10% glycerol and 0.2-0.5 MArginine or 0.2-0.5 M Arginine-HCl using 10 kDa cut off dialysis bag toremove imidazole. Size exclusion chromatography was performed ifnecessary as final polishing step using sephacryl 200 or superdex 200with automated chromatography system. Alternatively, urea denaturedprotein solution (of MEV-1) is either purified by IMAC purification andsubsequently or directly refolded by dialysis using 10 kDa dialysis bagin presence of refolding buffers with gradual decreasing ureaconcentration and ending at zero or negligible urea concentration,0.2-0.5 M Arginine or 0.2-0.5M Arginine-HCl, 10%-20% Glycerol. Therefolded recombinant protein MEV1 is purified by size exclusionchromatography as single step purification or if required as final stepof purification for IMAC purified protein using sephacryl 200 orsephadex 200 or superdex 200 with AKTA (automated chromatographicpurifier, GE). On the other hand, MEV2 protein was refolded indecreasing urea (from 6M to 0.1 M) and DTT (from 20 mM to negligibleconcentrations or 0.1 mM or even less) concentration in presence of0.2-1M Arginine or Arginine-HCL and/or redox pair reagents likeCysteine/Cystine or GSSG/GSH or oxidized DTT/reduced DTT orcystamine/cysteamine with concentrating between 0.05 mM to 10 mM. MEV2protein is purified with size exclusion chromatography as mentionedabove. Up to 96% pure protein of MEV2 can be achieved.

The protein was stored in polyols or sugars like 5-40% Glycerol or 5-60%sucrose or 5-40% Trehalose or 5-40% Sorbitol at −20° C. to (avoidrepeated freeze thaw which can be detrimental for the protein) protectfrom thermal stress given in Table 4.

Example 5: Results and Outcome of IB Protein MEV1 Extraction andPurification

The SDS-PAGE run of the eluted fractions from Ni-NTA columns (FIG. 5A)or direct purification by size exclusion chromatography showed thepresence of up to ˜94-96% respectively pure protein (Table 1 and 2).Western blot analysis with his tagged monoclonal antibody showed thespecific expression of the desired recombinant protein in the blot (FIG.5B).

TABLE 1 Purity grade from IMAC and Size exclusion chromatography(Applicable for MEV1) Step % age Purity After Lysis Between 22-28% AfterUrea Denaturation Between 50-60% After IMAC purification Between 90-94%purity After Size exclusion chromatography Between 92 upto 96% purity

TABLE 2 Purity Grade from Size exclusion chromatography Purification(Applicable for both MEV1 and MEV2) Step % age Purity After LysisBetween 22-28% After Urea Denaturation Between 50 60% After Sizeexclusion chromatography Purification Upto 96%

Example 6: Immunogenicity of MEV1 Recombinant Protein

All mice experiments were carried out strictly maintaining the procedurethat will be approved by the Animal Experiment Committee. Groups of6-week-old female BALB/c mice was inoculated with the recombinantprotein alone, in combination with Freund's complete adjuvant (CFA)Sigma), alum, MPLA and Alum, Ribi adjuvant, PolyIC, AddaVax (MF59 like)or PBS.

All mice were then boosted twice with the same dose in Freund'sincomplete adjuvant (IFA) or other respective adjuvants at a 2-weekinterval. Two-Three weeks after 1st immunization and/or two weeks after2^(nd) and 3^(rd) immunization, serum samples were prepared and storedfrozen until use. Animals were sacrificed after collecting the serum twoweeks after the last immunization.

ELISA plates were coated with 0.5-1 μg of MEV1 antigen or syntheticpeptides overnight in 0.1 M carbonate buffer (pH9.6). Next day, theplates were blocked for 1.5 hrs in 3% skimmed milk. Subsequently,serially diluted immunized sera were added in wells and incubated for1.5 hrs. After several washes with phosphate buffered saline withTween20 (PBST), the wells were incubated with 1:2000 diluted anti-mouseIgG HRP conjugated antibody (Thermo Scientific) or anti-mouse IgG1 HRPconjugated antibody (Thermo Scientific) or IgG2a HRP conjugated antibody(Abeam) and incubated for another 1 hr. After several washes with PBSTand final wash with PBS, TMB (3, 3′, 5, 5′-Tetraethylbenzidine)substrate for ELISA (Amresco) was added for color development andfinally the reaction was stopped with 0.6-1M HCL.

Animal immunogenicity studies performed in presence of differentadjuvants and subsequent analysis of the collected serum by ELISA hasshowed end-point titer ranging from 12500 to 100000 respectively inpresence of different adjuvants (FIG. 8) ( ). IgG1/IgG2a ratio greaterthan 1 depicts immune response predominantly associated with Th2lymphocytes and that of less than 1 denotes immune responsepredominantly associated with Th1 lymphocytes. The ratio of 12.04 wasobtained after immunization with 10 μg of antigen and CFA adjuvant asprime dose and antigen with IFA adjuvant combination as 2^(nd) boosterdose respectively (FIG. 6). The ratio was found to be 7.75 immunizedwith 10 μg of antigen with alum adjuvant as 2^(nd) booster dose (FIG.6). The antibody elicited is specific and highly immunogenic in presenceof alum and CFA/IFA adjuvants. The higher ratio of IgG1/IgG2a from theMEV1 immunized sera indicates the involvement of Th2 lymphocytes in theimmune response Immunization in presence of IFA has elicited strong IgG1response during terminal booster dose. MEV1 immunized sera in thepresence of alum has been found to direct more IgG1 immune responseindicating Th2 lymphocyte involvement that supports previous studieswhich have shown that alum is involved in Th2 associated immuneresponse. On the contrary 10 μg of antigen formulated with MPLA+ Alum,Poly(I:C) or AddaVax generated slight higher IgG2a with IgG1:IgG2a ratioof 0.47, 0.76 and 0.44 indicating overall balanced Th1/Th2 immuneresponse with slight biasness towards Th1 mediated immune response.

Inventors have also performed Interferon gamma (IFN-γ) and Interleukin-4(IL-4) quantitative assay which represents Th1 and Th2 lymphocytespecific immune response respectively from splenocytes isolated from themice immunized with MEV1 formulated with different adjuvants.Splenocytes were plated in the 24 well plate followed by stimulationwith antigen or peptides. After 72 hrs, supernatant were collected andcytokine assays were performed using kits. We have found thatrecombinant antigen formulated with MPLA and Alum is inducingsignificant Interferon gamma while the formulation with alum is inducingcomparatively more IL-4. Interferon gamma is the indicator for Th1 typeresponse while IL4 is the indicator for Th2 type immune response. Thus,the formulation in presence of MPLA and alum is inducing more Th1 typeimmune response and that with only alum is inducing more Th2 specificimmune response.

Immunofluorescence assay: Vero cells were infected with either EV71 orCVA16 virus at 0.1-0.2 MOI. One day post infection, the cells were fixedwith methanol-acetone solution (1:1v/v) for 1 hr. Then, the cells wereblocked with 1-3% BSA for 1 hr. Subsequently, the cells were incubatedwith serially diluted serum for 1.5 hrs at room temperature or overnightat 4° C. After several washes with PBS, the cells were incubated withanti-mouse IgG Alexa Fluor 488 conjugated antibody (Thermo Scientific)for 1 hr and observed under fluorescence microscope.

Immunofluorescence assay with virus infected and mock infected Vero cellusing serum raised against MEV1 as primary antibody showed that theantibody raised against MEV1 antigen binds with both EV71 and CVA16viruses (FIG. 7). EV71 antigen specific titer of 6400 and CVA16 antigenspecific titer of 800 is achieved with MEV1.

In vitro neutralization assay: In vitro neutralization was assessed by50% reduction in Plaque (PRNT50). Vero cells were infected withpre-optimized countable Plaque forming units (PFU) of virus or mixtureof virus and serially diluted sera (1:1) in 12 well format. After 1.5hrs post infection, the cells were overlayered with 0.8% Carboxymethylcellulose (CMC) (Sigma) in media. The cells were fixed in 10% formalinfour days post infection and stained with 0.8% crystal violet solutionfor plaque visualization. PRNT50 was calculated as the highest seradilution showing 50% or more reduction in the number of plaques.

It was found that MEV1 adjuvanted with MPLA and alum combination isshowing highest neutralization titer against EV71 and CVA16 (Table 3).

TABLE 3 PRNT50 titer Adjuvant Formulation EV71 CVA16 CFA/IFA 40 20 Alum30 15 Alum + MPLA 80 20 Adavax (MF59 like) 30 15 Poly(I:C) 30 15

The results obtained from immunofluorescence and PRNT₅₀ assay indicatedthe cross reactivity and cross-protectivity of the elicited antibodyagainst both the viruses. Whole IgG, IgG isotype assay and cytokineassays showed that MEV1 can generate both cellular and humoral immuneresponse significantly in present of appropriate adjuvants.

TABLE 4 Stability studies with purified synthetic MEV-1 Temp Time 4° C.(5 ± 3° C.) 37 ± 2° C. 1 week 95% >90% 2 week 95% >70% 1 month 95% 3month 93% 6 month 91%

1. A vaccine composition for prophylaxis against Hand Foot and MouthDisease caused by EV71 and CA16 comprising: a. vaccine antigen, the saidvaccine antigen is a synthetic construct selected from the recombinantprotein sequences as represented by SEQ ID No. 2 (named as MEV1) and SEQID No. 4 (named as MEV2); b. adjuvants; c. stabilizers; and d. aphysiologically acceptable buffer selected from phosphate and citrate;wherein the said vaccine formulation is stable for at least 2 years at5±3° C. and up to 2 weeks at 37° C.
 2. The vaccine composition asclaimed in claim 1, wherein the vaccine antigen having protein SEQ IDNo. 2 is obtained from a codon optimized gene sequence as represented bySEQ ID No.
 1. 3. The vaccine composition as claimed in claim 1, whereinthe vaccine antigen having protein SEQ ID No. 4 is obtained from a codonoptimized gene sequence as represented by SEQ ID No.
 3. 4. The vaccinecomposition as claimed in claim 1, wherein the adjuvant is selected froma group of alum (aluminium phosphate, aluminium hydroxide), squalenebased adjuvants such as MF59, montanide, RIM adjuvant, complete Freud'sadjuvant and incomplete Freud's, MPL, muramyl dipeptide, oil-in-wateremulsion, TLR ligand based adjuvants, CpG oligonucleotides, Non-CpGoligonucleotides, saponins such as QS-1, ISCOM, ISCOMATRIX, vitamins,and immunomodulants such as cytokines.
 5. The vaccine composition asclaimed in claim 1, wherein the stabilizers are selected from sugar5-60% sucrose or 5-40% Trehalose or sugar alcohols like 5-40% Glycerolor 5-40% Sorbitol.
 6. The vaccine composition as claimed in claim 1,wherein the said vaccine antigens SEQ ID No. 2 and SEQ ID No. 4 isprepared by a process comprising: a. expression of the protein sequencesSEQ ID No. 2 and SEQ ID No. 4 from codon optimized gene sequences,namely SEQ ID No. 1 and SEQ ID No. 3 respectively with 0.2-0.75 mM IPTGbetween 15° C.-37° C., in the form of inclusion bodies in a suitablehost selected from E. coli BL21 (DE3) cells or Rosetta cells; b.centrifugation at 10,000 rpm for 5 minutes to obtain the protein ofinterest in the cell pellet; c. suspending the pellet of step (b) withlysis buffer-I, the said lysis buffer-I for MEV1 comprising 50 mMNa₂HPO₄, 0.3M NaCl, 1% TritonX-114, 0.5 mg/ml lysozyme, 1 mM AEBSF,simultaneously performing freeze thaw cycles for at least 3 timesfollowed by ultrasonication for 20 seconds with at least 30 secondsintervals in between; d. alternatively undergoing lysis with lysisbuffer-I for MEV2, the said lysis buffer-I for MEV1 comprising LysisBuffer-I for MEV2 comprised 50 mM Tris, 0.3 M NaCl, 1% Triton X114, 0.5mg/ml lysozyme, 1 mM AEBSF at pH 8-8.5; e. undergoing cell lysis withlysis buffer-II for MEV1, the said lysis buffer-II for MEV1 comprising3-6M Urea, 5-10 mM imidazole, 50 mM Na₂HPO₄, 0.3M NaCl, and 1 mM AEBSFat 4° C. for 3-16 hours to recover the insoluble protein at pH of 7.4for MEV1 to obtain a denatured protein solution of MEV1; f.alternatively undergoing cell lysis with lysis buffer-II for MEV2, thesaid lysis buffer-II for MEV2 comprising 50 mM Tris, 0.3 M NaCl, 1 mMAEBSF in presence of 6M urea and 10-20 mM DTT; g. adding the denaturedprotein solution of step (e) above to Ni-NTA Immobilized Metal AffinityChromatography column followed by washing with wash buffer-I comprising50 mM Na₂HPO₄, 0.3M NaCl, 1 mM AEBSF, 3M urea at pH7.4 with 0.1% TritonX-114 and 5-10 mM Imidazole; h. again performing washing after step (g)above for MEV1 with Wash Buffer-II comprising 50 mM Na₂HPO₄, 0.3M NaCl,1 mM AEBSF, 3-6M urea at pH7.4 with 0.1% Triton X-114 and 20 mMimidazole; followed by washing by Wash Buffer-III, the said washbuffer-III comprising 50 mM Na₂HPO₄, 0.3 M NaCl, 1 mM AEBSF additionallywith 20 mM imidazole with decreasing concentrations of urea; i. elutingthe target protein MEV1 with elution buffer, the said elution buffercomprising 50 mM Na₂HPO₄, 0.3M NaCl, 1 mM AEBSF by increasingconcentrations of imidazole (250-500 mM) optionally with 3-6 M urea; j.undergoing dialysis of MEV-1 after step (i) above against phosphatebuffer saline using 10 kDa cutoff, membrane and refolding with refoldingbuffers comprising 0.1-0.3M NaCl, 10% glycerol and 0.2-0.5 M Arginine or0.2-0.5 M Arginine-HCl optionally followed by size exclusionchromatography to obtain the final protein MEV-1 with at least 92% to96% purity; k. undergoing refolding of MEV-2 after step (f) above indecreasing urea and DTT concentration in presence of 0.2-1M Arginine orArginine-HCL, redox pair reagents selected from Cysteine/Cystine,GSSG/GSH, oxidized DTT/reduced DTT or cystamine/cysteamine withconcentration between 0.05 mM to 10 mM to obtain the final protein MEV-2with at least 96% purity.
 7. A method of preparing the vaccine antigensSEQ ID No. 2 and SEQ ID No. 4 of claim 1 comprising the steps: a.expression of the protein sequences SEQ ID No. 2 and SEQ ID No. 4 fromcodon optimized gene sequences, namely SEQ ID No. 1 and SEQ ID No. 3respectively with 0.2-0.75 mM IPTG between 15° C.-37° C., in the form ofinclusion bodies in a suitable host selected from E. coli BL21 (DE3)cells or Rosetta cells; b. centrifugation at 10,000 rpm for 5 minutes toobtain the protein of interest in the cell pellet; c. suspending thepellet of step (b) with lysis buffer-I, the said lysis buffer-I for MEV1comprising 50 mM Na₂HPO₄, 0.3M NaCl, 1% TritonX-114, 0.5 mg/ml lysozyme,1 mM AEBSF, simultaneously performing freeze thaw cycles for at least 3times followed by ultrasonication for 20 seconds with at least 30seconds intervals in between; d. alternatively undergoing lysis withlysis buffer-I for MEV2, the said lysis buffer-I for MEV1 comprisingLysis Buffer-I for MEV2 comprised 50 mM Tris, 0.3 M NaCl, 1% TritonX114, 0.5 mg/ml lysozyme, 1 mM AEBSF at pH 8-8.5; e. undergoing celllysis with lysis buffer-II for MEV1, the said lysis buffer-II for MEV1comprising 3-6M Urea, 5-10 mM imidazole, 50 mM Na₂HPO₄, 0.3M NaCl, and 1mM AEBSF at 4° C. for 3-16 hours to recover the insoluble protein at pHof 7.4 for MEV1 to obtain a denatured protein solution of MEV1; f.alternatively undergoing cell lysis with lysis buffer-II for MEV2, thesaid lysis buffer-II for MEV2 comprising 50 mM Tris, 0.3 M NaCl, 1 mMAEBSF in presence of 6M urea and 10-20 mM DTT; g. adding the denaturedprotein solution of step (e) above to Ni-NTA Immobilized Metal AffinityChromatography column followed by washing with wash buffer-I comprising50 mM Na₂HPO₄, 0.3M NaCl, 1 mM AEBSF, 3M urea at pH7.4 with 0.1% TritonX-114 and 5-10 mM Imidazole; h. again performing washing after step (g)above for MEV1 with Wash Buffer-II comprising 50 mM Na₂HPO₄, 0.3M NaCl,1 mM AEBSF, 3-6M urea at pH7.4 with 0.1% Triton X-114 and 20 mMimidazole; followed by washing by Wash Buffer-III, the said washbuffer-III comprising 50 mM Na₂HPO₄, 0.3 M NaCl, 1 mM AEBSF additionallywith 20 mM imidazole with decreasing concentrations of urea; i. elutingthe target protein MEV1 with elution buffer, the said elution buffercomprising 50 mM Na₂HPO₄, 0.3M NaCl, 1 mM AEBSF by increasingconcentrations of imidazole (250-500 mM) optionally with 3-6 M urea; j.undergoing dialysis of MEV-1 after step (i) above against phosphatebuffer saline using 10 kDa cutoff, membrane and refolding with refoldingbuffers comprising 0.1-0.3M NaCl, 10% glycerol and 0.2-0.5 M Arginine or0.2-0.5 M Arginine-HCl optionally followed by size exclusionchromatography to obtain the final protein MEV-1 with at least 92% to96% purity; k. undergoing refolding of MEV-2 after step (f) above indecreasing urea (from 6 M to 0.1 M) and DTT (from 20 mM to 0.1 mM)concentration in presence of 0.2-1M Arginine or Arginine-HCL, redox pairreagents selected from Cysteine/Cystine, GSSG/GSH, oxidized DTT/reducedDTT or cystamine/cysteamine with concentration between 0.05 mM to 10 mMto obtain the final protein MEV-2 with at least 96% purity.
 8. Amulti-epitope synthetic protein sequence as disclosed in SEQ ID No. 2with at least 92%-96% purity.
 9. A multi-epitope synthetic proteinsequence as disclosed in SEQ ID no. 4 with at least 96% purity.